Temporal dynamics of immune-stromal cell interactions in fracture healing.

Bone fracture repair is a complex, multi-step process that involves communication between immune and stromal cells to coordinate the repair and regeneration of damaged tissue. In the US, 10% of all bone fractures do not heal properly without intervention, resulting in non-union. Complications from non-union fractures are physically and financially debilitating. We now appreciate the important role that immune cells play in tissue repair, and the necessity of the inflammatory response in initiating healing after skeletal trauma. The temporal dynamics of immune and stromal cell populations have been well characterized across the stages of fracture healing. Recent studies have begun to untangle the intricate mechanisms driving the immune response during normal or atypical, delayed healing. Various in vivo models of fracture healing, including genetic knockouts, as well as in vitro models of the fracture callus, have been implemented to enable experimental manipulation of the heterogeneous cellular environment. The goals of this review are to (1): summarize our current understanding of immune cell involvement in fracture healing (2); describe state-of-the art approaches to study inflammatory cells in fracture healing, including computational and in vitro models; and (3) identify gaps in our knowledge concerning immune-stromal crosstalk during bone healing.

Synovial macrophage diversity and activation of M-CSF signaling in post-traumatic osteoarthritis.

Objective: Synovium is home to immune and stromal cell types that orchestrate inflammation following a joint injury; in particular, macrophages are central protagonists in this process. We sought to define the cellular and temporal dynamics of the synovial immune niche in a mouse model of post-traumatic osteoarthritis (PTOA), and to identify stromal-immune crosstalk mechanisms that coordinate macrophage function and phenotype. Design: We induced PTOA in mice using a non-invasive tibial compression model of anterior cruciate ligament rupture (ACLR). Single cell RNA-seq and flow cytometry were used to assess immune cell populations in healthy (Sham) and injured (7d and 28d post-ACLR) synovium. Characterization of synovial macrophage polarization states was performed, alongside computational modeling of macrophage differentiation, as well as implicated transcriptional regulators and stromal-immune communication axes. Results: Immune cell types are broadly represented in healthy synovium, but experience drastic expansion and speciation in PTOA, most notably in the macrophage portion. We identified several polarization states of macrophages in synovium following joint injury, underpinned by distinct transcriptomic signatures, and regulated in part by stromal-derived macrophage colony-stimulating factor signaling. The transcription factors Pu.1, Cebpα, Cebpß, and Jun were predicted to control differentiation of systemically derived monocytes into pro-inflammatory synovial macrophages. Conclusions: We defined different synovial macrophage subpopulations present in healthy and injured mouse synovium. Nuanced characterization of the distinct functions, origins, and disease kinetics of macrophage subtypes in PTOA will be critical for targeting these highly versatile cells for therapeutic purposes.

Blood and adipose-resident eosinophils are defined by distinct transcriptional profiles.

Eosinophils are granular leukocytes of the innate immune system that play important functions in host defense. Inappropriate activation of eosinophils can occur in pathologies such as asthma and esophagitis. However, eosinophils also reside within adipose tissue, where they play homeostatic roles and are important in the activation of thermogenic beige fat. Here we performed bulk RNA sequencing in mouse adipose tissue-resident eosinophils isolated from both subcutaneous and gonadal depots, for the first time, and compared gene expression to blood eosinophils. We found a predominantly conserved transcriptional landscape in eosinophils between adipose depots that is distinct from blood eosinophils in circulation. Through exploration of differentially expressed transcription factors and transcription factors with binding sites enriched in adipose-resident eosinophil genes, we identified KLF, CEBP, and Fos/Jun family members that may drive functional specialization of eosinophils in adipose tissue. These findings increase our understanding of tissue-specific eosinophil heterogeneity, with implications for targeting eosinophil function to treat metabolic disorders such as obesity.

Inflammation in osteoarthritis: the latest progress and ongoing challenges.

PURPOSE OF REVIEW: The understanding of inflammation in osteoarthritis is rapidly evolving. This review highlights important basic science, mechanistic, and clinical findings since 2020 that underscore the current notion of osteoarthritis as an inflammatory disease. RECENT FINDINGS: There exists a disconnect between clinical radiographic findings and patient symptoms in osteoarthritis. Inflammation, in particular synovitis, has been put forward as a potential explanation for this disconnect. New findings have shed light on the temporal dynamics and activation states of joint-resident or systemically derived immune cell populations, notably macrophages, that participate in the inflammatory response. The intricate crosstalk in which they engage may underpin disparate pain and symptoms in patients, for instance during osteoarthritis flares. The role of biological and environmental factors such as exercise, age, and diet, have been the subject of recent studies for their protective or destructive roles in osteoarthritis inflammation. Despite these advances, no disease-modifying osteoarthritis treatments targeting inflammation have emerged. SUMMARY: Osteoarthritis is a debilitating chronic disease that manifests with widely varying symptomatology. Inflammation is now appreciated as a key pathophysiological process in osteoarthritis, but there remain considerable gaps in our understanding of its role in disease progression and how best to target the inflammatory response for therapeutic interventions.

Synovial fibroblasts assume distinct functional identities and secrete R-spondin 2 in osteoarthritis.

OBJECTIVES: Synovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/ß-catenin signalling, given its emerging importance in arthritis. METHODS: We subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain of function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types. RESULTS: We uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/ß-catenin signalling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4hi lining fibroblasts. Trajectory analyses predicted that Prg4hi lining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages and chondrocytes. CONCLUSIONS: Synovial fibroblasts assume distinct functional identities during PTOA in mice, and Prg4hi lining fibroblasts secrete Rspo2 that may drive pathological joint crosstalk after injury.

Acetylcholine-synthesizing macrophages in subcutaneous fat are regulated by ß2 -adrenergic signaling.

Non-neuronal cholinergic signaling, mediated by acetylcholine, plays important roles in physiological processes including inflammation and immunity. Our group first discovered evidence of non-neuronal cholinergic circuitry in adipose tissue, whereby immune cells secrete acetylcholine to activate beige adipocytes during adaptive thermogenesis. Here, we reveal that macrophages are the cellular protagonists responsible for secreting acetylcholine to regulate thermogenic activation in subcutaneous fat, and we term these cells cholinergic adipose macrophages (ChAMs). An adaptive increase in ChAM abundance is evident following acute cold exposure, and macrophage-specific deletion of choline acetyltransferase (ChAT), the enzyme for acetylcholine biosynthesis, impairs the cold-induced thermogenic capacity of mice. Further, using pharmacological and genetic approaches, we show that ChAMs are regulated via adrenergic signaling, specifically through the ß2 adrenergic receptor. These findings demonstrate that macrophages are an essential adipose tissue source of acetylcholine for the regulation of adaptive thermogenesis, and may be useful for therapeutic targeting in metabolic diseases.

The Heating Microenvironment: Intercellular Cross Talk Within Thermogenic Adipose Tissue.

Adipose tissue serves as the body's primary energy storage site; however, findings in recent decades have transformed our understanding of the multifaceted roles of this adaptable organ. The ability of adipose tissue to undergo energy expenditure through heat generation is termed adaptive thermogenesis, a process carried out by thermogenic adipocytes. Adipocytes are the primary parenchymal cell type in adipose tissue, yet these cells are sustained within a rich stromal vascular microenvironment comprised of adipose stem cells and progenitors, immune cells, neuronal cells, fibroblasts, and endothelial cells. Intricate cross talk between these diverse cell types is essential in regulating the activation of thermogenic fat, and the past decade has shed significant light on how this intercellular communication functions. This review will draw upon recent findings and current perspectives on the sophisticated repertoire of cellular and molecular features that comprise the adipose thermogenic milieu.

Eosinophil function in adipose tissue is regulated by Krüppel-like factor 3 (KLF3).

The conversion of white adipocytes to thermogenic beige adipocytes represents a potential mechanism to treat obesity and related metabolic disorders. However, the mechanisms involved in converting white to beige adipose tissue remain incompletely understood. Here we show profound beiging in a genetic mouse model lacking the transcriptional repressor Krüppel-like factor 3 (KLF3). Bone marrow transplants from these animals confer the beige phenotype on wild type recipients. Analysis of the cellular and molecular changes reveal an accumulation of eosinophils in adipose tissue. We examine the transcriptomic profile of adipose-resident eosinophils and posit that KLF3 regulates adipose tissue function via transcriptional control of secreted molecules linked to beiging. Furthermore, we provide evidence that eosinophils may directly act on adipocytes to drive beiging and highlight the critical role of these little-understood immune cells in thermogenesis.

Adrenergic-Independent Signaling via CHRNA2 Regulates Beige Fat Activation.

Maintaining energy homeostasis upon environmental challenges, such as cold or excess calorie intake, is essential to the fitness and survival of mammals. Drug discovery efforts targeting ß-adrenergic signaling have not been fruitful after decades of intensive research. We recently identified a new beige fat regulatory pathway mediated via the nicotinic acetylcholine receptor subunit CHRNA2. Here, we generated fat-specific Chrna2 KO mice and observed thermogenic defects in cold and metabolic dysfunction upon dietary challenges caused by adipocyte-autonomous regulation in vivo. We found that CHRNA2 signaling is activated after acute high fat diet feeding and this effect is manifested through both UCP1- and creatine-mediated mechanisms. Furthermore, our data suggested that CHRNA2 signaling may activate glycolytic beige fat, a subpopulation of beige adipocytes mediated by GABPα emerging in the absence of ß-adrenergic signaling. These findings reveal the biological significance of the CHRNA2 pathway in beige fat biogenesis and energy homeostasis.

Krüppel-like factor 3 (KLF3) suppresses NF-κB-driven inflammation in mice.

Bacterial products such as lipopolysaccharides (or endotoxin) cause systemic inflammation, resulting in a substantial global health burden. The onset, progression, and resolution of the inflammatory response to endotoxin are usually tightly controlled to avoid chronic inflammation. Members of the NF-κB family of transcription factors are key drivers of inflammation that activate sets of genes in response to inflammatory signals. Such responses are typically short-lived and can be suppressed by proteins that act post-translationally, such as the SOCS (suppressor of cytokine signaling) family. Less is known about direct transcriptional regulation of these responses, however. Here, using a combination of in vitro approaches and in vivo animal models, we show that endotoxin treatment induced expression of the well-characterized transcriptional repressor Krüppel-like factor 3 (KLF3), which, in turn, directly repressed the expression of the NF-κB family member RELA/p65. We also observed that KLF3-deficient mice were hypersensitive to endotoxin and exhibited elevated levels of circulating Ly6C+ monocytes and macrophage-derived inflammatory cytokines. These findings reveal that KLF3 is a fundamental suppressor that operates as a feedback inhibitor of RELA/p65 and may be important in facilitating the resolution of inflammation.

EoTHINophils: Eosinophils as key players in adipose tissue homeostasis.

Eosinophils are granular cells of the innate immune system that are found in almost all vertebrates and some invertebrates. Knowledge of their wide-ranging roles in health and disease has largely been attained through studies in mice and humans. Although eosinophils are typically associated with helminth infections and allergic diseases such as asthma, there is building evidence that beneficial homeostatic eosinophils residing in specific niches are important for tissue development, remodelling and metabolic control. In recent years, the importance of immune cells in the regulation of adipose tissue homeostasis has been a focal point of research efforts. There is an abundance of anti-inflammatory innate immune cells in lean white adipose tissue, including macrophages, eosinophils and group 2 innate lymphoid cells, which promote energy homeostasis and stimulate the development of thermogenic beige adipocytes. This review will evaluate evidence for the role of adipose-resident eosinophils in local tissue homeostasis, beiging and systemic metabolism, highlighting where more research is needed to establish the specific effector functions that adipose eosinophils perform in response to different internal and external cues.

Protein Arginine Methyltransferase 1 Interacts With PGC1α and Modulates Thermogenic Fat Activation.

Protein arginine methyltransferases (PRMTs) are enzymes that regulate the evolutionarily conserved process of arginine methylation. It has been reported that PRMTs are involved in many metabolic regulatory pathways. However, until now, their roles in adipocyte function, especially browning and thermogenesis, have not been evaluated. Even though Prmt1 adipocyte-specific-deleted mice (Prmt1fl/flAQcre) appeared normal at basal level, following cold exposure or ß-adrenergic stimulation, impaired induction of the thermogenic program was observed in both the interscapular brown adipose tissue and inguinal white adipose tissue of Prmt1fl/flAQcre mice compared with littermate controls. Different splicing variants of Prmt1 have been reported. Among them, PRMT1 variant 1 and PRMT1 variant 2 (PRMT1V2) are well conserved between humans and mice. Both variants contribute to the activation of thermogenic fat, with PRMT1V2 playing a more dominant role. Mechanistic studies using cultured murine and human adipocytes revealed that PRMT1V2 mediates thermogenic fat activation through PGC1α, a transcriptional coactivator that has been shown to play a key role in mitochondrial biogenesis. To our knowledge, our data are the first to demonstrate that PRMT1 plays a regulatory role in thermogenic fat function. These findings suggest that modulating PRMT1 activity may represent new avenues to regulate thermogenic fat and mediate energy homeostasis. This function is conserved in human primary adipocytes, suggesting that further investigation of this pathway may ultimately lead to therapeutic strategies against human obesity and associated metabolic disorders.

Characterization of Glucose Transporter 6 in Lipopolysaccharide-Induced Bone Marrow-Derived Macrophage Function.

The polarization processes for M1 versus M2 macrophages are quite distinct in the context of changes in cellular metabolism. M1 macrophages are highly glycolytic, whereas M2 macrophages require a more oxidative nutrient metabolism. An important part of M1 polarization involves upregulation of the glucose transporter (GLUT) GLUT1 to facilitate increased glucose uptake and glycolytic metabolism; however, the role of other glucose transporters in this process is largely unknown. In surveying the Functional Annotation of the Mammalian Genome and Gene Expression Omnibus Profiles databases, we discovered that the glucose transporter GLUT6 is highly upregulated in LPS-activated macrophages. In our previous work, we have not detected mouse GLUT6 protein expression in any noncancerous tissue; therefore, in this study, we investigated the expression and significance of GLUT6 in bone marrow-derived macrophages from wild-type and GLUT6 knockout C57BL/6 mice. We show that LPS-induced M1 polarization markedly upregulated GLUT6 protein, whereas naive macrophages and IL-4-induced M2 macrophages do not express GLUT6 protein. However, despite strong upregulation of GLUT6 in M1 macrophages, the absence of GLUT6 did not alter M1 polarization in the context of glucose uptake, glycolytic metabolism, or cytokine production. Collectively, these data show that GLUT6 is dispensable for LPS-induced M1 polarization and function. These findings are important because GLUT6 is an anticancer drug target, and this study suggests that inhibition of GLUT6 may not impart detrimental side effects on macrophage function to interfere with their antitumor properties.

Partial reprogramming of heterologous cells by defined factors to generate megakaryocyte lineage-restricted biomolecules.

The ability of transcriptional regulators to drive lineage conversion of somatic cells offers great potential for the treatment of human disease. To explore the concept of switching on specific target genes in heterologous cells, we developed a model system to screen candidate factors for their ability to activate the archetypal megakaryocyte-specific chemokine platelet factor 4 (PF4) in fibroblasts. We found that co-expression of the transcriptional regulators GATA1 and FLI1 resulted in a significant increase in levels of PF4, which became magnified over time. This finding demonstrates that such combinations can be used to produce potentially beneficial chemokines in readily available heterologous cell types.

Defining Eosinophil Function in Adiposity and Weight Loss.

Despite promising early work into the role of immune cells such as eosinophils in adipose tissue (AT) homeostasis, recent findings revealed that elevating the number of eosinophils in AT alone is insufficient for improving metabolic impairments in obese mice. Eosinophils are primarily recognized for their role in allergic immunity and defence against parasitic worms. They have also been detected in AT and appear to contribute to adipose homeostasis and drive energy expenditure, but the underlying mechanisms remain elusive. It has long been recognized that immune cells such as macrophages respond to external signals to regulate adipose homeostasis and energy balance, however, less is known about the relevance of eosinophil activity in AT. As the authors propose in this review, given recent debate over the relative importance of their tissue-specific abundance, the stage is now set for exploring the functionality and activation states of AT eosinophils.

Krüppel-like Factor 3 (KLF3/BKLF) Is Required for Widespread Repression of the Inflammatory Modulator Galectin-3 (Lgals3).

The Lgals3 gene encodes a multifunctional ß-galactoside-binding protein, galectin-3. Galectin-3 has been implicated in a broad range of biological processes from chemotaxis and inflammation to fibrosis and apoptosis. The role of galectin-3 as a modulator of inflammation has been studied intensively, and recent evidence suggests that it may serve as a protective factor in obesity and other metabolic disorders. Despite considerable interest in galectin-3, little is known about its physiological regulation at the transcriptional level. Here, using knockout mice, chromatin immunoprecipitations, and cellular and molecular analyses, we show that the zinc finger transcription factor Krüppel-like factor 3 (KLF3) directly represses galectin-3 transcription. We find that galectin-3 is broadly up-regulated in KLF3-deficient mouse tissues, that KLF3 occupies regulatory regions of the Lgals3 gene, and that KLF3 directly binds its cognate elements (CACCC boxes) in the galectin-3 promoter and represses its activation in cellular assays. We also provide mechanistic insights into the regulation of Lgals3, demonstrating that C-terminal binding protein (CtBP) is required to drive optimal KLF3-mediated silencing. These findings help to enhance our understanding of how expression of the inflammatory modulator galectin-3 is controlled, opening up avenues for potential therapeutic interventions in the future.

Phosphorylation of Krüppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity.

Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3.

Adipokines and insulin action: A sensitive issue.

Obesity is a major public health concern and a strong risk factor for insulin resistance, type 2 diabetes mellitus (T2DM), and cardiovascular disease. The last two decades have seen a reconsideration of the role of white adipose tissue (WAT) in whole body metabolism and insulin action. Adipose tissue-derived cytokines and hormones, or adipokines, are likely mediators of metabolic function and dysfunction. While several adipokines have been associated with obese and insulin-resistant phenotypes, a select group has been linked with insulin sensitivity, namely leptin, adiponectin, and more recently, adipolin. What is known about these insulin-sensitizing molecules and their effects in healthy and insulin resistant states is the subject of this review. There remains a significant amount of research to do to fully elucidate the mechanisms of action of these adipokines for development of therapeutics in metabolic disease.

Loss of Krüppel-like factor 3 (KLF3/BKLF) leads to upregulation of the insulin-sensitizing factor adipolin (FAM132A/CTRP12/C1qdc2).

Krüppel-like factor 3 (KLF3) is a transcriptional regulator that we have shown to be involved in the regulation of adipogenesis in vitro. Here, we report that KLF3-null mice are lean and protected from diet-induced obesity and glucose intolerance. On a chow diet, plasma levels of leptin are decreased, and adiponectin is increased. Despite significant reductions in body weight and adiposity, wild-type and knockout animals show equivalent energy intake, expenditure, and excretion. To investigate the molecular events underlying these observations, we used microarray analysis to compare gene expression in Klf3(+/+) and Klf3(-/-) tissues. We found that mRNA expression of Fam132a, which encodes a newly identified insulin-sensitizing adipokine, adipolin, is significantly upregulated in the absence of KLF3. We confirmed that KLF3 binds the Fam132a promoter in vitro and in vivo and that this leads to repression of promoter activity. Further, plasma adipolin levels were significantly increased in Klf3(-/-) mice compared with wild-type littermates. Boosting levels of adipolin via targeting of KLF3 offers a novel potential therapeutic strategy for the treatment of insulin resistance.

Holding Tight: Cell Junctions and Cancer Spread.

Cell junctions are sites of intercellular adhesion that maintain the integrity of epithelial tissue and regulate signalling between cells. These adhesive junctions are comprised of protein complexes that serve to establish an intercellular cytoskeletal network for anchoring cells, in addition to regulating cell polarity, molecular transport and communication. The expression of cell adhesion molecules is tightly controlled and their downregulation is essential for epithelial-mesenchymal transition (EMT), a process that facilitates the generation of morphologically and functionally diverse cell types during embryogenesis. The characteristics of EMT are a loss of cell adhesion and increased cellular mobility. Hence, in addition to its normal role in development, dysregulated EMT has been linked to cancer progression and metastasis, the process whereby primary tumors migrate to invasive secondary sites in the body. This paper will review the current understanding of cell junctions and their role in cancer, with reference to the abnormal regulation of junction protein genes. The potential use of cell junction molecules as diagnostic and prognostic markers will also be discussed, as well as possible therapies for adhesive dysregulation.